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Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl - levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P 2 depletion by activating phosphatidylinositol 5-phosphatases or G q protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P 2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P 2 -binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P 2 . Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P 2 ., (© 2024. The Author(s).)

TMEM16A is a Ca 2+ -activated Cl - channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP 2 in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl - currents, which are independent of the membrane potential and specific to PI(4,5)P 2 depletion. The attenuation of endogenous PI(4,5)P 2 levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl - currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P 2 , not PI4P and PI(3,4,5)P 3 . Finally, we also confirmed that PI(4,5)P 2 resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P 2 selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle., (© 2020 Myeong et al.)

Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) sensitivity of voltage-gated Ca 2+ (Ca V ) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca V 2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca V 2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca V 2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall Ca V channel gating properties.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. Proton sensing by ASICs has been known to mediate pain, mechanosensation, taste transduction, learning and memory, and fear. In this study, we investigated the differential subcellular localization of ASIC2a and ASIC3 in heterologous expression systems. While ASIC2a targeted the cell surface itself, ASIC3 was mostly accumulated in the ER with partial expression in the plasma membrane. However, when ASIC3 was co-expressed with ASIC2a, its surface expression was markedly increased. By using bimolecular fluorescence complementation (BiFC) assay, we confirmed the heteromeric association between ASIC2a and ASIC3 subunits. In addition, we observed that the ASIC2a-dependent surface trafficking of ASIC3 remarkably enhanced the sustained component of the currents. Our study demonstrates that ASIC2a can increase the membrane conductance sensitivity to protons by facilitating the surface expression of ASIC3 through herteromeric assembly. [BMB Reports 2016; 49(10): 542-547].

Voltage-gated calcium (Cav) channels, which are regulated by membrane potential, cytosolic Ca(2+), phosphorylation, and membrane phospholipids, govern Ca(2+) entry into excitable cells. Cav channels contain a pore-forming α1 subunit, an auxiliary α2δ subunit, and a regulatory β subunit, each encoded by several genes in mammals. In addition to a domain that interacts with the α1 subunit, β2e and β2a also interact with the cytoplasmic face of the plasma membrane through an electrostatic interaction for β2e and posttranslational acylation for β2a. We found that an increase in cytosolic Ca(2+) promoted the release of β2e from the membrane without requiring substantial depletion of the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane. Experiments with liposomes indicated that Ca(2+) disrupted the interaction of the β2e amino-terminal peptide with membranes containing PIP2 Ca(2+) binding to calmodulin (CaM) leads to CaM-mediated inactivation of Cav currents. Although Cav2.2 coexpressed with β2a required Ca(2+)-dependent activation of CaM for Ca(2+)-mediated reduction in channel activity, Cav2.2 coexpressed with β2e exhibited Ca(2+)-dependent inactivation of the channel even in the presence of Ca(2+)-insensitive CaM. Inducible depletion of PIP2 reduced Cav2.2 currents, and in cells coexpressing β2e, but not a form that lacks the polybasic region, increased intracellular Ca(2+) further reduced Cav2.2 currents. Many hormone- or neurotransmitter-activated receptors stimulate PIP2 hydrolysis and increase cytosolic Ca(2+); thus, our findings suggest that β2e may integrate such receptor-mediated signals to limit Cav activity., (Copyright © 2016, American Association for the Advancement of Science.)

Voltage-gated Ca(2+) (CaV) channels are dynamically modulated by G protein-coupled receptors (GPCR). The M1 muscarinic receptor stimulation is known to enhance CaV2.3 channel gating through the activation of protein kinase C (PKC). Here, we found that M1 receptors also inhibit CaV2.3 currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated CaV2.3 currents by ∼two-fold. After the PMA-induced potentiation, stimulation of M1 receptors decreased the CaV2.3 currents by 52 ± 8%. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is responsible for the muscarinic suppression of CaV2.3 currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of PI(4,5)P2 to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed CaV2.3 currents, by 53 ± 3%. Next, dephosphorylation of both PI(4)P and PI(4,5)P2 to PI by PJ translocation further decreased the current by up to 66 ± 3%. The results suggest that CaV2.3 currents are modulated by the M1 receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane PI(4,5)P2. Our results also suggest that there is rapid turnover between PI(4)P and PI(4,5)P2 in the plasma membrane.

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca(V)2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation., (© 2015 Kim et al.)

The β-subunits of voltage-gated Ca(2+) (Ca(V)) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca(V) channels. We find that Ca(V) β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). When Ca(V)1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP(2). When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP(2) sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca(V) β-subunits on PIP(2) regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca(V) β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca(V)2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca(V) channel inhibition by PIP(2) depletion. Evidently lipidation and membrane anchoring of Ca(V) β-subunits compete with the PIP(2) regulation of high-voltage-activated Ca(V) channels. Compared with expression with Ca(V) β3-subunits alone, inhibition of Ca(V)2.2 channels by PIP(2) depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca(V) currents in neurons would be regulated by membrane PIP(2) to a degree that depends on their endogenous β-subunit combinations.

Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) from the plasma membrane. We give evidence that Mg(2+) and polyamines weaken the KCNQ channel-phospholipid interaction. Lowering internal Mg(2+) augmented inward and outward KCNQ currents symmetrically, and raising Mg(2+) reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (>>+6). The inhibitory effects of Mg(2+) were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K(+) currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Igamma to accelerate synthesis of PIP(2) attenuated the sensitivity to polyvalent cations. We suggest that Mg(2+) and other polycations reduce the currents by electrostatic binding to the negative charges of PIP(2), competitively reducing the amount of free PIP(2) available for interaction with channels. The dose-response curves could be modeled by a competition model that reduces the pool of free PIP(2). This mechanism is likely to modulate many other PIP(2)-dependent ion channels and cellular processes.

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and are turned off when muscarinic receptors stimulate cleavage of PIP(2) by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP(2) and diacylglycerol in tsA cells. The cells are transfected with M(1) muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.

Phosphatidylinositol 4,5-bisphosphate is a signaling phospholipid of the plasma membrane that has a dynamically changing concentration. In addition to being the precursor of inositol trisphosphate and diacylglycerol, it complexes with and regulates many cytoplasmic and membrane proteins. Recent work has characterized the regulation of a wide range of ion channels by phosphatidylinositol 4,5-bisphosphate, helping to redefine the role of this lipid in cells and in neurobiology. In most cases, phosphatidylinositol 4,5-bisphosphate increases channel activity, and its hydrolysis by phospholipase C reduces channel activity.

Mastoparan, a peptide toxin from wasp venome, mimics receptors by stimulating the GTPase activity of guanine nucleotide binding proteins and the G-protein-coupled phospholipase C (PLC). By using Mas-7, the active analog of mastoparan, we showed that it makes pores in the plasma membrane. Treatment with Mas-7 but not Mas-17, the inactive analog, produced a concentration-dependent rise in intracellular Ca2+ concentration ([Ca2+]i) and facilitated the uptake of ethidium bromide (EtBr) (314 Da) to a sustained level during the stimulation. In addition, Mas-7 triggered the influx of lucifer yellow (457 Da), while it did not induce the influx of fura-2 (831 Da) and Evans blue (961 Da). However, the Mas-7-induced permeability was selectively prevented by the addition of La3+, Ni2+, and Co2+, but not Cd2+. This blocking activity was concentration-dependent. While the stimulatory effect of Mas-7 on PLC activity was dependent on extracellular Ca2+, the pore forming activity of Mas-7 was not effected by removal of extracellular Ca2+. These results, therefore, suggest that the mastoparan's action in pore formation is independent from its action in PLC stimulation and is negatively effected by inorganic cations.